A-band Titin Research Articles

Generation of four distinct isogenic cell lines with truncating variants in I-band or A-band titin

Truncating variants in TTN (TTNtv) are present in 15–25 % of patients with idiopathic dilated cardiomyopathy. Interestingly, the pathogenicity of TTNtv seems to be linked to their location within the gene. More proximal I-band TTNtv (TTNtvI) harbour less pathogenic potential than distant A-band TTNtv (TTNtvA). We created isogenic human induced pluripotent stem cell lines (hiPSC) with TTNtvI and TTNtvA using CRISPR/Cas9, for the investigation of the pathomechanism in hiPSC-derived cardiomyocytes (hiPSC-CMs). Exon 48 (E48), located in the I-band, and exon 357 (E357), located in the A-band were targeted.

Molecular insights into titin’s A-band

The thick filament-associated A-band region of titin is a highly repetitive component of the titin chain with important scaffolding properties that support thick filament assembly. It also has a demonstrated link to human disease. Despite its functional significance, it remains a largely uncharacterized part of the titin protein. Here, we have performed an analysis of sequence and structure conservation of A-band titin, with emphasis on poly-FnIII tandem components. Specifically, we have applied multi-dimensional sequence pairwise similarity analysis to FnIII domains and complemented this with the crystallographic elucidation of the 3D-structure of the FnIII-triplet A84-A86 from the fourth long super-repeat in the C-zone (C4). Structural models serve here as templates to map sequence conservation onto super-repeat C4, which we show is a prototypical representative of titin’s C-zone. This templating identifies positionally conserved residue clusters in C super-repeats with the potential of mediating interactions to thick-filament components. Conservation localizes to two super-repeat positions: Ig domains in position 1 and FnIII domains in position 7. The analysis also allows conclusions to be drawn on the conserved architecture of titin’s A-band, as well as revisiting and expanding the evolutionary model of titin’s A-band.

Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 μm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 μM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

The assembly and evaluation of antisense oligonucleotides applied in exon skipping for titin-based mutations in dilated cardiomyopathy

The leading cause of genetic dilated cardiomyopathy (DCM) is due to mutations in the TTN gene, impacting approximately 15–20% of familial and 18% of sporadic DCM cases. Currently, there is potential for a personalized RNA-based therapeutic approach in titin-based DCM, utilizing antisense oligonucleotide (AON) mediated exon-skipping, which attempts to reframe mutated titin transcripts, resulting in shortened, functional protein. However, the TTN gene is massive with 363 exons; each newly identified TTN exon mutation provides a challenge to address when considering the potential application of AON mediated exon skipping. In the initial phase of this strategy, the mutated TTN exon requires specific AON design and evaluation to assess the exon skipping effectiveness for subsequent experiments.Here, we present a detailed protocol to effectively assemble and evaluate AONs for efficient exon-skipping in targeted TTN exons. We chose a previously identified TTN 1-bp deletion mutation in exon 335 as an exemplary target exon, which causes a frameshift mutation leading to truncated A-band titin in DCM. We designed two specific AONs to mask the Ttn exon 335 and confirmed successfully mediated exon skipping without disrupting the Ttn reading frame. In addition, we evaluated and confirmed AON-treated HL-1 cells show maintained store-operated calcium entry, fractional shortening as well as preserved sarcomeric formation in comparison to control samples, indicating the treated cardiomyocytes retain adequate, essential cell function and structure, proving the treated cells can compensate for the loss of exon 335. These results indicate our method offers the first systematic protocol in designing and evaluating AONs specifically for mutated TTN target exons, expanding the framework of future advancements in the therapeutic potential of antisense-mediated exon skipping in titin-based DCM.

A-Band Titin Truncation in Zebrafish Causes Dilated Cardiomyopathy and Hemodynamic Stress Intolerance

Background Truncating variants in the TTN gene ( TTNtv) are common in patients with dilated cardiomyopathy (DCM) but also occur in the general population. Whether TTNtv are sufficient to cause DCM or require a second hit for DCM manifestation is an important clinical issue. Methods We generated a zebrafish model of an A-band TTNtv identified in 2 human DCM families in which early-onset disease appeared to be precipitated by ventricular volume overload. Cardiac phenotypes were serially assessed from 0 to 12 months using video microscopy, high-frequency echocardiography, and histopathologic analysis. The effects of sustained hemodynamic stress resulting from an anemia-induced hyperdynamic state were also evaluated. Results Homozygous ttna mutants had severe cardiac dysmorphogenesis and premature death, whereas heterozygous mutants ( ttnatv/+) survived into adulthood and spontaneously developed DCM. Six-month-old ttnatv/+ fish had reduced baseline ventricular systolic function and failed to mount a hypercontractile response when challenged by hemodynamic stress. Pulsed wave and tissue Doppler analysis also revealed unsuspected ventricular diastolic dysfunction in ttnatv/+ fish with prolonged isovolumic relaxation and increased diastolic passive stiffness in the absence of myocardial fibrosis. These defects reduced diastolic reserve under stress conditions and resulted in disproportionately greater atrial dilation than observed in wild-type fish. Conclusions Heterozygosity for A-band titin truncation is sufficient to cause DCM in adult zebrafish. Abnormalities of systolic and diastolic reserve in titin-truncated fish reduce stress tolerance and may contribute to a substrate for atrial arrhythmogenesis. These data suggest that hemodynamic stress may be an important modifiable risk factor in human TTNtv-related DCM.

Topology of interaction between titin and myosin thick filaments.

Titin is a giant protein spanning between the Z- and M-lines of the sarcomere. In the A-band titin is associated with the myosin thick filament. It has been speculated that titin may serve as a blueprint for thick-filament formation due to the super-repeat structure of its A-band domains. Accordingly, titin might provide a template that determines the length and structural periodicity of the thick filament. Here we tested the titin ruler hypothesis by mixing titin and myosin at in situ stoichiometric ratios (300 myosins per 12 titins) in buffers of different ionic strength (KCl concentration range 100-300 mM). The topology of the filamentous complexes was investigated with atomic force microscopy. We found that the samples contained distinct, segregated populations of titin molecules and myosin thick filaments. We were unable to identify complexes in which myosin molecules were regularly associated to either mono- or oligomeric titin in either relaxed or stretched states of the titin filaments. Thus, the electrostatically driven self-association is stronger in both myosin and titin than their binding to each other, and it is unlikely that titin functions as a geometrical template for thick-filament formation. However, when allowed to equilibrate configurationally, long myosin thick filaments appeared with titin oligomers attached to their surface. The titin meshwork formed on the thick-filament surface may play a role in controlling thick-filament length by regulating the structural dynamics of myosin molecules and placing a mechanical limit on the filament length.

A.P.2: Proteomic profile of cytoplasmic bodies (CB) compared to non-CB aggregates in HMERF associated with mutations in A-band titin

Hereditary myopathy with early respiratory failure (HMERF) is caused by A-band titin mutations and characterized by myofibrillar alterations and protein aggregation in skeletal muscle fibers. Our previous studies revealed that the composition of myotilin-positive aggregates is comparable to that seen in myofibrillar myopathies (MFM). However, there are two different types of protein aggregates in HMERF: subsarcolemmal cytoplasmic bodies (CB) with a dense filamentous core and non-CB aggregates. The aim of this study was to compare the proteomic profile of these protein deposits. We used Gomori trichrome stained skeletal muscle sections from two HMERF patients to separately collect CB, non-CB aggregates and intraindividual control samples by laser microdissection and performed a highly sensitive label-free mass spectrometry approach for protein detection and relative quantification based on spectral counting. The proteomic profiles of CB and non-CB aggregates showed a high degree of similarity: XIRP2, desmin, filamin C, N-RAP, nestin, COL6A3, myotilin, sarcosin, αB-crystallin, dystrophin, XIN and aciculin ranked among the most abundant over-represented proteins (ratio >1.5 compared to control samples) in both aggregate types. Peptides assigned to these twelve proteins accounted for 69% (CB) and 75% (non-CB aggregates) of all peptides assigned to over-represented proteins and the individual proportions of these proteins were almost the same. We also detected similarities in less abundant over-represented proteins. Titin was under-represented in CB (ratio 0.81) and in non-CB aggregates (ratio 0.80). In conclusion, our analysis revealed that the proteomic profiles of CB and non-CB aggregates are highly similar in HMERF, especially regarding abundant over-represented proteins, and typical of MFM aggregates.

A.P.7: Misfolding of fibronectin III 119 subdomain in titin results in hereditary myopathy with early respiratory failure

Hereditary myopathy with early respiratory failure (HMERF) is a rare disease with muscle weakness and respiratory failure as early symptoms. Muscle pathology is characterized by the presence of multiple cytoplasmic bodies and other protein aggregates in muscle fibers. These protein aggregates are composed of multiple proteins including titin, aB-crystallin, actin, myotilin, desmin and others. Previous reports have described mutations in A-band titin ( TTN ) gene associated with HMERF. All patients harbor mutations located in exon 343 in the TTN gene that codes for the fibronectin III domain 119 (FN3 119) in the 10th motif of the 11-element motif A-band super-repeat. Based on the pathological findings in HMERF of protein aggregates in muscle fibers, we investigated if disease-causing mutations affect the biochemical behavior of this titin domain. All five disease-causing amino acid changes analyzed by us (p.P30068R, p.C30071R, p.W30088R, p.W30088C and p.P30091L) resulted in impaired FN3 119 domain solubility. In contrast, amino acid changes associated with common SNPs (p.V30076I, p.R30107C and p.S30125F) did not have this effect. In silico analyses further support the notion that disease-causing mutations impair proper folding of the FN3 119 domain. Our results suggest that HMERF is caused by defective protein folding. We speculate that the FN3 119 domain could be important for protein–protein interaction that could explain why all identified mutations causing HMERF are located in this particular domain.

Atypical phenotypes in titinopathies explained by second titin mutations

Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe, or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in 8 patients of 7 European families. Clinical, histopathological, and muscle imaging data of patients and family members were reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of 6 patients, 3 with an limb-girdle muscular dystrophy type 2J (LGMD2J) phenotype. Novel frameshift mutations were identified in 5 patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in 1 patient, and 1 Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation. The unequal expression levels of TTN transcripts in 5 probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism explaining phenotype variability.

Interactive Properties of A-Band Titin

The giant protein titin (chain weight ∼3 MDa) has important roles in assembly and contractile function in vertebrate striated muscle sarcomeres. The molecule consists principally of ∼300 Ig and Fn3 domains in a chain of more than 1 μm long spanning half the sarcomere. The A-band part is attached to the thick (myosin) filament with six molecules in each half filament. The I-band part makes an elastic connection between the tip of the filament and the Z-disc. The central region of A-band titin (C-zone, ∼0.5 μm long) contains 11 copies of what is called the large super-repeat, Ig-Fn3-Fn3-Ig-Fn3-Fn3-Fn3-Ig-Fn3-Fn3-Fn3. The lengths of the large super-repeat (∼43 nm) and of its sub-periods (∼14 nm) correspond to the two main periodicities of the thick filament. C-zone titin is thought to make at least three distinct types of interactions: with itself, with myosin, and with myosin binding protein-C. We studied three recombinant 2-domain fragments from the three sub-periods of a large super-repeat. At physiological ionic strength all three constructs were soluble and mostly monomeric, whereas reduced salt resulted in self-association. Self-association was strongest in the case of the third sub-period construct leading to its complete sedimentation. Electron microscopy revealed large net-like oligomers suggesting regularity in the domains interactions. In the constructs from the first two sub-periods, side-by-side dimers were observed and oligomers were seen only rarely. Co-sedimentation and solid phase assays showed binding of the constructs to the light meromyosin part of myosin but did not reveal any significant differences in the interactions. Modelling of the proteins folds and electrostatics was consistent with the observed interactions being mainly electrostatic.

O.4 Proteomic analysis confirms that HMERF associated with mutations in A-band titin is a new subtype of myofibrillar myopathies

Hereditary myopathy with early respiratory failure (HMERF) caused by A-band TTN mutations shows a clinical and histopathological overlap with myofibrillar myopathies (MFM) characterized by disintegration of myofibrils and protein aggregation in muscle fibers. We applied a proteomic approach to decipher the aggregate composition in HMERF and compared the proteomic profile with findings in MFM subtypes. Nine skeletal muscle samples from HMERF patients with three different mutations in A-band titin and histopathological findings consistent with MFM were analyzed. Protein aggregates and intraindividual control samples (from aggregate-free muscle fibers) were collected by laser microdissection and analysed by a label-free mass spectrometric approach for identification and relative quantification of proteins. Fifty-eight proteins showed a statistically significant accumulation in aggregates with a ratio > 1.8 compared to control samples. The over-represented proteins desmin, filamin C, Xirp2, N-RAP, alphaB-crystallin, nestin, myotilin, Xin and Hsp27 were highly abundant in aggregates. The same was found in different MFM subtypes. The detection of further Z-disc proteins, chaperones, proteins involved in protein degradation and sarcolemmal proteins was also typical of MFM. In addition, we identified an over-representation of proteins that are involved in signaling pathways (e.g. inhibitors of the Rho/ROCK signaling pathway and ankyrin repeat domain-containing proteins). Titin was slightly but significant under-represented in aggregates (ratio 0.83). In conclusion, the proteomic profile of aggregates in HMERF associated with mutations in A-band titin is typical of MFM and confirms that this type of titinopathy is a new MFM subtype. The finding that titin is under-represented in aggregates indicates that aggregate formation is mediated by secondary effects. The detected over-representation of proteins involved in signaling pathways may play a role in pathogenesis.

P.15.8 Hereditary myopathy with early respiratory failure (HMERF) – Rapid increase of identified titinopathy families worldwide

Hereditary myopathy with early respiratory failure (HMERF) was described in 7 families from Sweden in 1990. One titin mutation in the kinase domain of titin gene in two of these families was identified 2005, but the further understanding of the disease remained elusive despite a few reports on families with somewhat similar phenotypes. This has now changed completely: within one year 6 new papers have reported the molecular genetic resolution in 27 different families with a worldwide distribution and occurrence in different ethnic populations: Caucasian, Japanese and Native American. This explosion of detailed clarification of HMERF was possible due to identification of a pathognomonic pattern of muscle involvement on MRI and, above all, due to the use of exome sequencing to clarify the molecular genetic cause. All 27 families have mutations in one single exon 343 of titin gene encoding the fibronectin-like FN3 119 domain (formerly A150) of A-band titin. The most common mutation, also surprisingly identified in different populations, is a C30071R change showing sharing of alleles on a common haplotype in most of the families. One of the new mutations showed semi-recessive inheritance pattern with subclinical myopathy in the heterozygous parents. Typical clinical features were respiratory failure at mid-adulthood in an ambulant patient with very variable degree of muscle weakness. The pathognomonic muscle MRI pattern shows most severe fatty degenerative changes in semitendinosus, obturatorius, gracilis, tibialis posterior and anterior. Cytoplasmic bodies reactive for thin filament and Z-disc proteins combined with rimmed vacuoles constitute the muscle pathology hallmarks. This extraordinary evolution of understanding HMERF disease indicates that the disease is much more common than previously thought and that exon 343 of TTN is the primary target for molecular diagnosis.

Hereditary myopathy with early respiratory failure: occurrence in various populations

ObjectiveSeveral families with characteristic features of hereditary myopathy with early respiratory failure (HMERF) have remained without genetic cause. This international study was initiated to clarify epidemiology and the genetic underlying...

G.O.2 Hereditary myopathy with early respiratory failure associated with a mutation in A-band titin

Hereditary myopathy with early respiratory failure (HMERF) and extensive myofibrillar lesions have been described in sporadic and familial cases and linked to various chromosomal regions. We describe the clinical manifestations, muscle histopathology and genetics in eight individuals from three apparently unrelated families with clinical and pathological features of HMERF. All patients had muscle weakness in the pelvic girdle, neck flexors, respiratory and trunk muscles, and the majority had prominent calf hypertrophy. Examination of pulmonary function showed decreased vital capacity. No signs of cardiac muscle involvement were found. Muscle histopathological features included marked muscle fibre size variation, fibre splitting, numerous internal nuclei and fatty infiltration. Frequent groups of fibres showed eosinophilic inclusions and deposits. At the ultrastructural level there were extensive myofibrillar lesions with marked Z-disc alterations. Whole exome sequencing in four individuals from one family revealed a missense mutation, g.274375, T>C; p.Cys30071Arg, in the titin gene, TTN. The mutation, which changes a highly conserved residue in the myosin binding A-band titin, was demonstrated to segregate with the disease in all three families. High density single nucleotide polymorphism arrays covering the entire genome demonstrated sharing of a 699 Mb haplotype, located in chromosome region 2q31 including TTN, indicating common ancestry of this novel and first disease-causing mutation in A-band titin associated with HMERF.

Hereditary myopathy with early respiratory failure associated with a mutation in A-band titin

Hereditary myopathy with early respiratory failure and extensive myofibrillar lesions has been described in sporadic and familial cases and linked to various chromosomal regions. The mutated gene is unknown in most cases. We studied eight individuals, from three apparently unrelated families, with clinical and pathological features of hereditary myopathy with early respiratory failure. The investigations included clinical examination, muscle histopathology and genetic analysis by whole exome sequencing and single nucleotide polymorphism arrays. All patients had adult onset muscle weakness in the pelvic girdle, neck flexors, respiratory and trunk muscles, and the majority had prominent calf hypertrophy. Examination of pulmonary function showed decreased vital capacity. No signs of cardiac muscle involvement were found. Muscle histopathological features included marked muscle fibre size variation, fibre splitting, numerous internal nuclei and fatty infiltration. Frequent groups of fibres showed eosinophilic inclusions and deposits. At the ultrastructural level, there were extensive myofibrillar lesions with marked Z-disc alterations. Whole exome sequencing in four individuals from one family revealed a missense mutation, g.274375T>C; p.Cys30071Arg, in the titin gene (TTN). The mutation, which changes a highly conserved residue in the myosin binding A-band titin, was demonstrated to segregate with the disease in all three families. High density single nucleotide polymorphism arrays covering the entire genome demonstrated sharing of a 6.99 Mb haplotype, located in chromosome region 2q31 including TTN, indicating common ancestry. Our results demonstrate a novel and the first disease-causing mutation in A-band titin associated with hereditary myopathy with early respiratory failure. The typical histopathological features with prominent myofibrillar lesions and inclusions in muscle and respiratory failure early in the clinical course should be incentives for analysis of TTN mutations.

1H, 15N and 13C backbone chemical shift assignment of the titin A67-A68 domain tandem

Single molecules of the giant protein titin extend across half of the muscle sarcomere, from the Z-line to the M-line, and have roles in muscle assembly and elasticity. In the A-band titin is attached to thick filaments and here the domain arrangement occurs in regular patterns of eleven called the large super-repeat. The large super-repeat itself occurs eleven times and forms nearly half the titin molecule. Interactions of the large super-repeats with myosin are consistent with a role in thick filament assembly. Here we report backbone assignments of the titin A67-A68 domain tandem (Fn-Ig) from the third super-repeat (A65-A75) completed using triple resonance NMR experiments.

Structural Studies of an Immunoglobulin-Fibronectin Type III Domain Tandem from Titin

Titin is a giant multidomain protein in muscle. Single molecules span half the sarcomere, from the Z-disk to the M-band, and are involved in sarcomere assembly and elasticity. In the A-band titin is attached to thick filaments and here the domain arrangement is in regular patterns of eleven, called large super-repeats. The large super-repeat occurs eleven times and forms nearly half the titin molecule. Interactions of the large super-repeats with myosin and C-protein suggest a role in thick filament assembly.

The Structure of the FnIII Tandem A77-A78 Points to a Periodically Conserved Architecture in the Myosin-Binding Region of Titin

Titin is a large intrasarcomeric protein that, among its many roles in muscle, is thought to modulate the in vivo assembly of the myosin motor filament. This is achieved through the molecular template properties of its A-band region, which is composed of fibronectin type III (FnIII) and immunoglobulin (Ig) domains organized into characteristic 7-domain (D-zone) and 11-domain (C-zone) superrepeats. Currently, there is little knowledge on the structural details of this region of titin. Here we report the conformational characterization of three FnIII tandems, A77-A78, A80-A82, and A84-A86, which are components of the representative fourth C-zone superrepeat. The structure of A77-A78 has been elucidated by X-ray crystallography to 1.65 Å resolution, while low-resolution models of A80-A82 and A84-A86 have been calculated using small-angle X-ray scattering. A77-A78 adopts an extended “up–down” domain arrangement, where domains are connected by a hydrophilic three-residue linker sequence. The linker is embedded in a rich network of polar contacts at the domain interface that results in a stiff molecular conformation. The models of A80-A82 and A84-A86, which contain hydrophobic six-residue-long interdomain linkers, equally showed elongated molecular shapes, but with slightly coiled or zigzagged conformations. Small-angle X-ray scattering data further suggested that the long linkers do not result in a noticeable increase in molecular flexibility but lead to semibent domain arrangements. Our findings indicate that the structural characteristics of FnIII tandems from A-band titin contrast markedly with those of poly-Ig tandems from the elastic I-band, which exhibit domain interfaces depleted of interactions and compliant conformations. Furthermore, the analysis of sequence conservation in FnIII domains from A-band titin points to the existence of conformationally defined interfaces at specific superrepeat positions, possibly leading to a periodic and locally ordered architecture supporting the molecular scaffold properties of this region of titin.

Cardiac Titin

Titin constitutes the third myofilament of cardiac muscle, with a single giant polypeptide spanning from Z-disk to the M-band region of the sarcomere1 (Fig. 1). The ∼1.0 MDa region in the I-band is extensible and consists of tandemly arranged immunoglobulin (Ig)-like domains that make up proximal (near Z-disk) and distal (near A-I junction) segments, interspersed by the PEVK sequence (rich in proline, glutamate, valine, and lysine residues) and the N2B element2. Each functions as a distinct spring element3. The C-terminal ∼2 MDa of titin is located in the A-band and is inextensible. It is composed of regular arrays of Ig and fibronectin type 3 (Fn3) modules forming so-called super-repeats2. A-band titin may function as a molecular ruler, regulating assembly of the thick filament2,4,5. Titin's ∼250-kDa COOH-terminal region is an integral part of the M-band and contains a kinase domain6,7. As in the Z-disk, where titin filaments from opposite sarcomeres overlap, titin filaments from opposite half-sarcomeres overlap within the M-band, where they are interconnected by M-band proteins8. Thus, titin filaments with opposite polarity overlap in both Z-disk and M-band, forming a contiguous filament along the myofibril. In this review, we discuss titin's functions in the heart, with an emphasis on its role in diastolic function and the various mechanisms whereby passive stiffness can be tuned. Due to space constraints, it has not been possible to provide inclusive references to all original articles in the field. Figure 1 Schematic of titin in sarcomere. Differential splicing Titin is encoded by a single gene containing 368 exons. Multiple splice pathways in the I-band encoding region (∼230 exons) give rise to isoforms with different spring composition9. The three cardiac isoform classes are shown in Fig. 1. The relatively small ∼3.0 MDa isoform is known as N2B titin (it contains the N2B element)9. A second class also contains the N2A element, and is termed N2BA titin. N2BA titins have a longer PEVK segment and a variable number of additional Ig domains resulting in a ∼3.3-3.5 MDa size9. The third class includes isoforms that predominate in fetal-neonatal life which contain additional spring elements in both tandem Ig and PEVK regions, resulting in a ∼3.6-3.8 MDa size protein10-12. These isoforms gradually disappear during postnatal development. Regulation of the spring composition of titin in fetal-neonatal myocardium allows adjustment of diastolic filling properties during development.

Structural Investigation of a Fibronectin Type III Domain Tandem from the A-band of the Titin

Single molecules of the giant muscle protein titin span half muscle sarcomeres, from the Z-disk to the M-band, and have key roles in sarcomere assembly and elasticity. In the A-band titin is attached to thick filaments and here the sequence shows fibronectin type III and immunoglobulin-like domains. These are mostly arranged in regular patterns of eleven domains called the large super-repeats. The large super-repeat occurs eleven times and this entire region thus forms nearly half of the titin molecule. Through interactions with myosin and C-protein, it is involved in thick filament assembly. We are determining the atomic structure, dynamical properties and the inter-domain arrangement of overlapping double and triple domain fragments of the large super-repeat by NMR spectroscopy. Ultimately, we hope to combine the data to reconstruct the overall conformation of the super-repeat. Here we investigated the A59-A60 domain tandem, which was expressed in bacteria from cDNA. The assignment of the backbone atoms was obtained using triple resonance NMR experiments. An initial structure was determined by backbone chemical shifts and homology modeling using the CS23D and Rosetta software packages. It was refined using RDC data to give realistic models for both domains. As we expected, these are both double-β-sheet sandwich structures characteristic of fibronectin type III domains. We are also carrying out relaxation measurements to probe the dynamics of the domains and their linker region.